The decrease of cell responsiveness to a persistent stimulus, usually termed desensitization, is a widespread biological phenomenon. The signaling by G protein-coupled receptors (GPCRs) is attenuated by a two-step mechanism: receptor phosphorylation by a specific kinase followed by arrestin binding to active phosphoreceptor. Arrestin binding terminates G protein-mediated signaling, tags GPCRs for internalization, and redirects signaling to other pathways via non-receptor binding partners (c-Src and MAP kinases, ubiquitin ligases, etc). Arrestins also interact with microtubules via the same interface that is involved in receptor binding and mobilize several signaling molecules to the cytoskeleton with different functional consequences. The main objective of this proposal is to elucidate the structural basis of arrestin function as an organizer of multi-protein signaling complexes in the cell. We hypothesize that the signaling capability of arrestin molecule is determined by its conformation. We propose to identify arrestin elements involved in receptor and microtubule binding and the nature of receptor- and microtubule-induced conformational changes in both non- visual arrestins that regulate their interactions with non-receptor partners: kinases c-Src, ERK2, JNK3, ubiquitin ligase Mdm2, etc. To this end, we propose to use site-directed mutagenesis, direct binding assay, site-directed spin labeling of arrestins and EPR spectroscopy, as well as functional assays in living cells. We also propose to use double spin-labeled arrestins and spin-labeled arrestins with spin-labeled model receptor, rhodopsin, to measure inter-spin distances in arrestin-receptor complex by conventional EPR and double electron-electron resonance (DEER) to obtain a working structural model of the complex. This information will set the stage for designing arrestin-based molecular tools for targeted manipulation of cellular signaling that can be used for experimental and therapeutic purposes. PUBLIC HEALTH RELEVANCE: Arrestins are multi-functional adaptors that mobilize various signaling molecules to G protein-coupled receptors and microtubules with different functional consequences. The goal of this proposal is to elucidate the conformations of receptor-bound and microtubule-bound arrestins to understand how arrestin conformation affects its interactions with signaling proteins and the consequences of their binding. This information will set the stage for designing arrestin-based molecular tools for targeted manipulation of cellular signaling that can be used for experimental and therapeutic purposes.